Iron-sulfur (Fe-S) clusters are protein cofactors that play essential roles in virtually every facet of cellular physiology ranging from nucleic acid metabolism to aerobic respiration to the metabolism of nucleotides, amino acids, and lipids. The assembly and transfer of these cofactors to cytoplasmic and nuclear substrates is carried out by the cytoplasmic Fe-S protein assembly (CIA) machinery, a highly conserved biosynthetic pathway devoted to their biogenesis. Despite its central role in this process, our understanding of the CIA pathway remains lacking. My laboratory recently discovered a novel `CIA targeting complex' consisting of MMS19, FAM96B, and CIAO1 that functions at a late stage in the CIA pathway to recruit distinct sets of apoproteins to the CIA machinery and facilitate their assembly. In the proposed work, we will use a combination of biochemistry and proteomic mass spectrometry to investigate fundamental aspects of CIA targeting complex function. In specific aim 1, we will focus on understanding how the CIA targeting complex recognizes substrates. We will utilize both hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cross-linking mass spectrometry (CX-MS) approaches to globally map protein interactions between the CIA targeting complex and its substrates in order to understand how molecular recognition and specificity are achieved. Specific aim 2 will examine the role of subcellular localization in the regulation of CIA targeting complex function. We find that the CIA targeting complex is localized to the ER-Golgi and now seek to understand the molecular basis, function, and significance of this localization. Investigation of these two aims will elucidate fundamental features regarding the regulation and function of the CIA targeting complex while potentially offering novels insights into how dysregulation of Fe-S cluster assembly contributes to a wide range of neurological, hematological, and metabolic disorders.